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One of the main causes of poor prognoses in patient with glioblastoma (GBM) is drug resistance to current standard treatment, which includes chemoradiation and adjuvant temozolomide (TMZ). In addition, the concept of cancer stem cells provides new insights into therapy resistance and management also in GBM and glioblastoma stem cell-like cells (GSCs), which might contribute to therapy resistance. Bone morphogenetic protein-4 (BMP4) stimulates astroglial differentiation of GSCs and thereby reduces their self-renewal capacity. Exposure of GSCs to BMP4 may also sensitize these cells to TMZ. A recent phase I trial has shown that local delivery of BMP4 is safe, but a large variation in survival is seen in these treated patients and in features of their cultured tumors. We wanted to combine TMZ and BMP4 (TMZ + BMP4) therapy and assess the inter-tumoral variability in response to TMZ + BMP4 in patient-derived GBM cultures. A phase II trial could then benefit a larger group of patients than those treated with BMP4 only. We first show that simultaneous treatment with TMZ + BMP4 is more effective than sequential treatment. Second, when applying our optimized treatment protocol, 70% of a total of 20 GBM cultures displayed TMZ + BMP4 synergy. This combination induces cellular apoptosis and does not inhibit cell proliferation. Comparative bulk RNA-sequencing indicates that treatment with TMZ + BMP4 eventually results in decreased MAPK signaling, in line with previous evidence that increased MAPK signaling is associated with resistance to TMZ. Based on these results, we advocate further clinical trial research to test patient benefit and validate pathophysiological hypothesis. [ABSTRACT FROM AUTHOR]

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents. Author summary: Seoul orthohantavirus is a rat-borne zoonotic virus that can cause hemorrhagic fever with renal syndrome in humans worldwide while it generally leads to persistent infection in absence of clinical signs in the natural reservoir host, the Norway rat. It is believed that persistent infection in reservoir hosts is due to downregulation of pro-inflammatory immune responses and upregulation of regulatory responses, while in humans excessive immune responses contribute to disease. Endothelial cells in the lungs are of particular interest as they represent a primary target during Seoul orthohantavirus infection in both species. So far, it remains incompletely understood how the responses of these cells to Seoul orthohantavirus contribute to the development of disease in humans and the prevention of such in rats. In this study, we compared the host responses of human and rat lung endothelial cells to Seoul orthohantavirus infection and found that anti-viral responses from both species are comparable, but differ in timing. Additionally, we identified a novel protein interaction network during later phases of acute infection in rat cells, which may play a crucial role in the development of disease in humans and persistence in rodents. [ABSTRACT FROM AUTHOR]

Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably. [ABSTRACT FROM AUTHOR]

Puumala orthohantavirus (PUUV) infection in humans can result in hemorrhagic fever with renal syndrome. Endothelial cells (ECs) are primarily infected with increased vascular permeability as a central aspect of pathogenesis. Historically, most studies included ECs cultured under static two-dimensional (2D) conditions, thereby not recapitulating the physiological environment due to their lack of flow and inherent pro-inflammatory state. Here, we present a high-throughput model for culturing primary human umbilical vein ECs in 3D vessels-on-chip in which we compared host responses of these ECs to those of static 2D-cultured ECs on a transcriptional level. The phenotype of ECs in vessels-on-chip more closely resembled the in vivo situation due to higher similarity in expression of genes encoding described markers for disease severity and coagulopathy, including IDO1 , LGALS3BP , IL6 and PLAT , and more diverse endothelial-leukocyte interactions in the context of PUUV infection. In these vessels-on-chip, PUUV infection did not directly increase vascular permeability, but increased monocyte adhesion. This platform can be used for studying pathogenesis and assessment of possible therapeutics for other endotheliotropic viruses even in high biocontainment facilities.

Astrocytes are essential for the formation and maintenance of neural networks. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques, primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing, we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons, consistent with their further maturation. In coculture with human neurons, multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min -1 (hPSC-derived), 2.86 ± 0.64 min -1 (rat); p  = 0.19]. In comparison, we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived), 24.77 ± 4.04 Hz (rat); p  < 0.01], and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived), 1.07 ± 0.14 Hz (rat); p  < 0.001], consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived), 8.39 ± 0.63/100 μm 2 (rat); p  < 0.001]. Taken together, we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation, providing a fully human platform for investigating astrocyte function and neuronal-glial interactions., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Lendemeijer et al.)

Noncoding RNAs have been widely recognized as essential mediators of gene regulation. However, in contrast to protein-coding genes, much less is known about the influence of noncoding RNAs on human diseases. Here we examined the association of genetic variants located in primary microRNA sequences and long noncoding RNAs (lncRNAs) with Alzheimer disease (AD) by leveraging data from the largest genome-wide association meta-analysis of late-onset AD. Variants annotated to 5 miRNAs and 10 lncRNAs (in seven distinct loci) exceeded the Bonferroni-corrected significance threshold (p < 1.02 × 10 -6 ). Among these, a leading variant (rs2526377:A>G) at the 17q22 locus annotated to two noncoding RNAs (MIR142 and BZRAP1-AS) was significantly associated with a reduced risk of AD and fulfilled predefined criteria for being a functional variant. Our functional genomic analyses revealed that rs2526377 affects the promoter activity and decreases the expression of miR-142. Moreover, differential expression analysis by RNA-Seq in human iPSC-derived neural progenitor cells and the hippocampus of miR-142 knockout mice demonstrated multiple target genes of miR-142 in the brain that are likely to be involved in the inflammatory and neurodegenerative manifestations of AD. These include TGFBR1 and PICALM, of which their derepression in the brain due to reduced expression levels of miR-142-3p may reduce the risk of AD., (© 2019 Wiley Periodicals, Inc.)

Background: Despite high heritability, little success was achieved in mapping genetic determinants of depression-related traits by means of genome-wide association studies., Methods: To identify genes associated with depressive symptomology, we performed a gene-based association analysis of nonsynonymous variation captured using exome-sequencing and exome-chip genotyping in a genetically isolated population from the Netherlands (n = 1999). Finally, we reproduced our significant findings in an independent population-based cohort (n = 1604)., Results: We detected significant association of depressive symptoms with a gene NKPD1 (p = 3.7 × 10 -08 ). Nonsynonymous variants in the gene explained 0.9% of sex- and age-adjusted variance of depressive symptoms in the discovery study, which is translated into 3.8% of the total estimated heritability (h 2 = 0.24). Significant association of depressive symptoms with NKPD1 was also observed (n = 1604; p = 1.5 × 10 -03 ) in the independent replication sample despite little overlap with the discovery cohort in the set of nonsynonymous genetic variants observed in the NKPD1 gene. Meta-analysis of the discovery and replication studies improved the association signal (p = 1.0 × 10 -09 )., Conclusions: Our study suggests that nonsynonymous variation in the gene NKPD1 affects depressive symptoms in the general population. NKPD1 is predicted to be involved in the de novo synthesis of sphingolipids, which have been implicated in the pathogenesis of depression., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.

Background: ECHS1 encodes a mitochondrial enzyme involved in the degradation of essential amino acids and fatty acids. Recently, ECHS1 mutations were shown to cause a new severe metabolic disorder presenting as Leigh or Leigh-like syndromes. The objective of this study was to describe a family with 2 siblings affected by different dystonic disorders as a resulting phenotype of ECHS1 mutations., Methods: Clinical evaluation, MRI imaging, genome-wide linkage, exome sequencing, urine metabolite profiling, and protein expression studies were performed., Results: The first sibling is 17 years old and presents with generalized dystonia and severe bilateral pallidal MRI lesions after 1 episode of infantile subacute metabolic encephalopathy (Leigh-like syndrome). In contrast, the younger sibling (15 years old) only suffers from paroxysmal exercise-induced dystonia and has very mild pallidal MRI abnormalities. Both patients carry compound heterozygous ECHS1 mutations: c.232G>T (predicted protein effect: p.Glu78Ter) and c.518C>T (p.Ala173Val). Linkage analysis, exome sequencing, cosegregation, expression studies, and metabolite profiling support the pathogenicity of these mutations. Expression studies in patients' fibroblasts showed mitochondrial localization and severely reduced levels of ECHS1 protein. Increased urinary S-(2-carboxypropyl)cysteine and N-acetyl-S-(2-carboxypropyl)cysteine levels, proposed metabolic markers of this disorder, were documented in both siblings. Sequencing ECHS1 in 30 unrelated patients with paroxysmal dyskinesias revealed no further mutations., Conclusions: The phenotype associated with ECHS1 mutations might be milder than reported earlier, compatible with prolonged survival, and also includes isolated paroxysmal exercise-induced dystonia. ECHS1 screening should be considered in patients with otherwise unexplained paroxysmal exercise-induced dystonia, in addition to those with Leigh and Leigh-like syndromes. Diet regimens and detoxifying agents represent potential therapeutic strategies. © 2016 International Parkinson and Movement Disorder Society., (© 2016 International Parkinson and Movement Disorder Society.)

The aim of our study is to investigate whether single-nucleotide dystrophin gene (DMD) variants associate with variability in cognitive functions in healthy populations. The study included 1240 participants from the Erasmus Rucphen family (ERF) study and 1464 individuals from the Rotterdam Study (RS). The participants whose exomes were sequenced and who were assessed for various cognitive traits were included in the analysis. To determine the association between DMD variants and cognitive ability, linear (mixed) modeling with adjustment for age, sex and education was used. Moreover, Sequence Kernel Association Test (SKAT) was used to test the overall association of the rare genetic variants present in the DMD with cognitive traits. Although no DMD variant surpassed the prespecified significance threshold (P<1 × 10(-4)), rs147546024:A>G showed strong association (β = 1.786, P-value = 2.56 × 10(-4)) with block-design test in the ERF study, while another variant rs1800273:G>A showed suggestive association (β = -0.465, P-value = 0.002) with Mini-Mental State Examination test in the RS. Both variants are highly conserved, although rs147546024:A>G is an intronic variant, whereas rs1800273:G>A is a missense variant in the DMD which has a predicted damaging effect on the protein. Further gene-based analysis of DMD revealed suggestive association (P-values = 0.087 and 0.074) with general cognitive ability in both cohorts. In conclusion, both single variant and gene-based analyses suggest the existence of variants in the DMD which may affect cognitive functioning in the general populations.

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods.